The most effective but largely ignored target for prostate cancer early detection and intervention

Over the past two decades, the global efforts for the early detection and intervention of prostate cancer seem to have made significant progresses in the basic researches, but the clinic outcomes have been disappointing: (1) prostate cancer is still the most common non-cutaneous cancer in Europe in men, (2) the age-standardized prostate cancer rate has increased in nearly all Asian and African countries, (3) the proportion of advanced cancers at the diagnosis has increased to 8.2% from 3.9% in the USA, (4) the worldwide use of PSA testing and digital rectal examination have failed to reduce the prostate cancer mortality, and (5) there is still no effective preventive method to significantly reduce the development, invasion, and metastasis of prostate cancer… Together, these facts strongly suggest that the global efforts during the past appear to be not in a correlated target with markedly inconsistent basic research and clinic outcomes. The most likely cause for the inconsistence appears due to the fact that basic scientific studies are traditionally conducted on the cell lines and animal models, where it is impossible to completely reflect or replicate the in vivo status. Thus, we would like to propose the human prostate basal cell layer (PBCL) as “the most effective target for the early detection and intervention of prostate cancer”. Our proposal is based on the morphologic, immunohistochemical and molecular evidence from our recent studies of normal and cancerous human prostate tissues with detailed clinic follow-up data. We believe that the human tissue-derived basic research data may provide a more realistic roadmap to guide the clinic practice and to avoid the potential misleading from in vitro and animal studies.

Together, these facts suggest that global efforts during the past appear to be not in a correlated target. The most likely cause appears due to the fact that basic scientific studies are traditionally conducted on the cell lines and animal models, which are impossible to completely reflect or replicate the in vivo status. Thus, we propose the prostate basal cell layer (PBCL) as "the most effective target for the early detection and intervention of prostate cancer" for the following reasons.

The PBCL is an essential component of the prostate gland
The human prostate gland consists of three components: the epithelium, capsule, and stroma. The epithelium is completely encircled by the capsule (a single layer of PBCL) and the basement membrane (BM, a thin sheet of extracellular fibers), and the stroma. Therefore, prostate cancer cells have to first pass through the PBCL and then BM to invade to the stroma [45][46][47][48][49] (Figure 1). Formalin-fixed and paraffin-embedded human prostate gland tissue sections were double immune-stained, 1A and 1B with antibodies to smooth muscle actin (SMA) that is reactive to the basement membrane (BM), smooth muscle, and endothelial cells, and CK34βE12, which is reactive to PBCL; 1C and 1D with SMA plus p63, which is reactive to PBCL. EP = Epithelium.

The focal PBCL degeneration is a triggering factor for tumor progression
The detailed dynamic cellular kinetics of the human prostate basal cell population remains elusive [55][56][57][58][59][60][61]. However, distinct senescence or degenerative changes of PBCL are frequently seen in some of normal and malignant prostate tissues. These changes include: focal disruptions (defined as the absence of basal cells resulting in a gap larger than the combined size of at least three basal cells in at least two or more consecutive sections), the loss of nuclear p63 expression, nuclear swelling, cell debris, and large clusters of cell debris. Luminal cells overlying focally disrupted PBCL often show markedly higher cellular density, proliferation rate, and morphology compared to those from their counterparts overlying nondisrupted PBCL (Figure 3).   expression; Thin arrows identify the residual PBCL; Red stars identify invasive lesions. Please note that the size of the focal disruptions in PBCL varies substantially, and that the epithelial cells overlying focally disrupted PBCL are morphologically more similar to the adjacent invasive cancer cells with a higher proliferation rate than their adjacent counterparts still enclosed by the residual PBCL.

The focal PBCL disruption is absolutely needed for tumor invasion
Our previous studies have consistently demonstrated that a focal disruption in a given PBCL is absolutely needed for the initiation of cancer invasion [62][63][64][65][66][67][68]. If the surrounding FBCL is intact, such tumors can grow to a very large size, but may remain at non-invasive state for many years ( Figure  4A and 4B). In a sharp contrast, if the surrounding FBCL is focally disrupted, the cancer invasion could commence at a very early stage, despite a very small size ( Figure 4C-4F). Double immunochemical staining with the cell proliferation specific marker Ki-67 and the PBCL specific marker CK34βE12 reveals that all invasion starts at the site of focal PBCL disruptions and a vast majority of proliferating cells are located at or near the site of focal PBCL disruptions. The "budding cells" from focally disrupted PBCL are often immediately adjacent to or in a direct continuity with the invasive cancer cells ( Figure 4C-4F). Selective gene expression profiling of microdissected human prostate gland tissue samples has consistently shown that cell clusters overlying focally disrupted PBCL have a significantly higher expression level of cell growth-and invasion-related molecules, including growth factors, stem cell lineage markers, anti-apoptosis-related genes, and endothelial cell makers than their morphologically similar counterparts enclosed by the residual PBCL [66] ( Figure 5).

The PBCL itself is the target of a variety of pathologic alterations
It has been consistently documented that prostate basal cell carcinoma is very rare, accounting for only about 0.01% of the total prostate malignancies [69 -73], which strongly suggests that the prostate basal cell population is very unlikely to be a major target of the prostate carcinogenesis. However, it has also been consistently demonstrated that the PBCL belongs to a self-renewal cell population [74][75][76][77][78][79][80]. For this and other reasons, the PBCL population itself also frequently suffers from a variety of degeneration-and regeneration-related pathologic alterations that includes, but is not limited to the following:

The loss or significant reduction of nuclear p63 expression in normal appearing PBCL
In autopsy, biopsy, and surgically resected human prostate gland tissues, a vast majority of the basal cells in normal or hyperplastic tissues are not only morphologically distinct, but also express a high level of p63 and Maspin, two well-documented tumor suppressors ( Figure 6A-6B). However, about 6% to 8% of the cases harbor variable numbers of morphologically normal appearing basal cell clusters that are completely devoid of, or have a significantly reduced cells with p63 nuclear expression ( Figure 6C-6D). Prostate cancer patients with such atypical basal cell clusters have a significantly more aggressive clinical courses and worse prognosis [81].

The loss of the expression of all PBCL specific markers
About 6% to 8% of the autopsy, biopsy, and surgically resected human prostate tissues also harbor a variable number of atypical pre-invasive cancer cell clusters, in which all the basal cell layers are largely non-disrupted and morphologically distinct, whereas they completely lack the expression of CK34βE12, p63, and Maspin. They are even completely devoid of the expression of the proliferating cell nuclear antigen (PCNA). These pre-invasive cancer cells enclosed by such PBCL are morphologically and immunohistochemically similar to adjacent invasive cancer cells, and have a significantly higher proliferation rate than their adjacent counterparts at the same stage [81] ( Figure 7).

Frequent apoptosis-related alterations
Distinct apoptosis-related alterations of basal cells are frequently seen in focally disrupted PBCL. Immunohistochemistry-based apoptotic assays clearly demonstrate that a vast majority of focally degenerated basal cells show high levels of apoptosis-related molecules. All apoptotic basal cells are physically located at or near focally disrupted PBCL. Epithelial cells adjacent to apoptotic basal cells have a much higher cell density than their adjacent counterparts ( Figure 8).

The significant infiltration of immune cells
Degenerated basal cell products appear to act as cytokines to attract infiltration of immune-reactive cells to the physical sites of focally disrupted PBCL, and infiltrated lymphocytes are generally associated with cell debris or morphologically degenerated basal cells (Figure 9).

Figure 8. Apoptosis-related alterations in degenerated basal cells.
Formalin-fixed and paraffin-embedded human prostate gland tissue sections were subjected to the apoptotic assays with a commercially available detection kit, and then, were immune-stained for CK34βE12 to elucidate PBCL. Arrows identify PBCL. Squares identify apoptotic basal cells. Circles identify epithelial cells overlying or near the focally disrupted PBCL.

The high level of Tenascin C expression in focally disrupted PBCL
Tenascin C is an extracellular matrix glycoprotein, which paves the paths and facilitates the migration and metastasis of prostate cancer cells [82][83][84][85][86]. Our previous studies have shown that Tenascin C is highly expressed at the site of distinct degenerative basal cells, and epithelial cells in the vicinity of areas with elevated Tenascin C often lose the cohesion [66] (Figure 10).
Based on above findings, it is apparent that PBCL is not only an essential constitute of the prostate gland, but also an active producer of tumor suppressors, which exert significant decisive influence on adjacent epithelial and stromal cells. In addition, as the PBCL population belongs to a self-renewal population, it has to consistently undergo normal self-replenishment processes to replace aged and injured cells. Consequently, the PBCL population itself also suffers from a wide variety of degeneration-and regeneration-related normal and pathological alterations.
The detailed cellular and molecular mechanisms of focal disruptions or a total loss of the PBCL remains elusive. Furthermore, there is no solid evidence to determine whether the loss of basal cells is a direct trigger for the development of prostate adenocarcinoma, or the loss of basal cells is directly resulted from prostate cancer cells. However, it has been consistently concluded that focal disruptions or a total loss of the PBCL is statistically correlated with the invasion and metastasis of almost all types of prostate cancer (except prostate basal cell carcinoma) [62][63][64][65][66][67][68][69][70][71][72][73].
Based on above facts, the PBCL appears to be the most effective but largely ignored target for the early detection and intervention of prostate cancer. As the morphologic, pathological, and immuno-histochemical profiles of the basal cell population is far more easily recognizable and definable than its epithelial counterpart, the PBCL appears to be a more easily readable roadmap with the following specific scientific and clinic implications and applications: • To use Maspin and PSA as independent risk factors for cancer screening. As Maspin is consistently expressed in basal cells [87][88][89][90], while the PSA is elevated in virtually all prostate malignancies [91][92][93][94], Maspin-and PSA-related signatures in the serum can be utilized for a population-based screening for the early detection of prostate cancer. • To use p63 as a risk factor for a population-based screening to detect the predisposition of cancer susceptibility or tumor suppressing genes. Since p63 belongs to the p53 tumor suppressor family, and is normally expressed in the nucleus of the basal cells [95,96], an aberrant expression level or subcellular localization of p63 accompanying by an elevated PSA level may signify the predisposition of cancer susceptibility or mutated suppressing genes. Previous studies have clearly demonstrated that loss or cytoplasmic expression of p63 is associated with elevated cancer stem cells, enhanced cell migration and metastasis, and increased mortality in prostate cancer [97][98][99].
• To use p63, Maspin, and different RNA signatures as biomarkers for the non-invasive detection of early prostate cancer. Previous studies have revealed that p63, Maspin, RNA signatures, and PSA are detectable in the urine samples of prostate cancer patients [100][101][102][103][104][105][106]. Thus, a statistical comparison of the expression levels of p63, Maspin, and PSA in the urine samples may be used as a non-invasive clinic test for the detection of early prostate cancer.
• To use exfoliated basal cells combined with other urinary markers for early detection of prostate cancer progression and invasion. Previous studies have consistently shown that a variable number of exfoliated cancer cells are detectable in a majority of prostate cancer patients [107][108][109][110].
As the basal cells are localized at the base of the epithelial cell layer, the detection of exfoliated basal cells in the urine is likely to signify the disruptions of the PBCL and the stromal invasion of the prostate cancer.
• To use the PBCL physical integrity (disrupted vs non-disrupted) combined with PSA test results as a clinic marker for the differentiation diagnosis. As the disruption of the PBCL is a prerequisite for prostate cancer invasion and metastasis, while an elevated PSA level is detectable in both non-invasive and invasive prostate cancer, the physical integrity of the PBCL in patients with an elevated PSA could effectively differentiate between non-invasive and invasive prostate cancer.
• To use the expression of Tenascin in PBCL as a routine clinic test of the prostate biopsy or urine sample. Previous studies have consistently demonstrated that aberrant Tenascin expression is exclusively seen at or near focally disrupted PBCL and is also significantly correlated with prostate cancer invasion and metastasis [66,[82][83][84][85][86]. Thus, the assessment of the PBCL-associated Tenascin expression may lead to the identification of the specific cases at increased risk for prostate cancer progression.
• To use Maspin or CK34βE12 as biomarkers to discriminate prostate from non-prostate cancers.
Previous studies have shown that (a) high levels of PSA is seen in patients with breast, lung, ovary, liver, kidney, adrenal, skin, salivary, and colorectal cancer [111][112][113], (b) smoking, asymptomatic inflammation, metformin use, chronic prostatitis can elevate the PSA expression level [114][115][116][117], and (c) age, ethnicity, triglyceride level, and BMI can also significantly impact the expression of PSA [118,119]. In contrast, above factors have little impact on PBCL. Thus, normal expression status of Maspin or CK34βE12 in individuals with high levels of PSA may signify non-prostate lesions.
• To use focal PBCL disruptions as a localizer to identify cancer-stem cell clusters/specific precursors of invasive cancer. Our previous studies of multiple cancers have consistently shown that a focal disruptions of tumor capsules selectively facilitate clonal proliferation of overlying cancer stem cells to form distinct cell clusters. These newly formed clusters have significantly higher levels of cancer stem cell markers and invasion and metastasis-related genes than their morphologically comparable counters still enclosed by the non-disrupted tumor capsules [62][63][64][65][66][120][121][122][123] (Figure 4 & 5). It is very likely that these cell clusters may represent the direct precursors of invasive lesions. Thus, micro-dissecting these clusters for further evaluation can potentially lead to the dentification of triggering molecules for the basal cell degenerations, tumor progression, and invasion.
• To use anti-inflammatory drug aspirin or statin to repair the PBCL degeneration-related tumor capsule disruptions. Previous studies have consistently shown that aspirin or statin could significantly alter the immune milieu of prostate and to prevent cancer progression [132][133][134]. Therefore, the administration of aspirin or statin to individuals with focally disrupted PBCL associated with significant infiltration of the immune cells (as shown in Figure 9) and chronic prostatitis could potentially reduce the extent of associated immune cells and facilitate the repairing of focally disrupted tumor capsules.
• To administer stem cell specific molecules, inducers, or stimulators to burst the normal replenishment and the physical integrity of PBCL. Previous studies have suggested that BMP5, Zeb1, CD24, CD44, NANOG, and Nestin are prostate stem cell-specific markers that are essential for the maintenance of the normal replenishment and physical integrity of the PBCL [135][136][137]. Thus, the administration of these molecules or stem cell specific inducers or stimulators to patients at a high risk of prostate cancer progression may offer the promise of more effective approaches for prostate cancer early intervention.
• To use basal cells lacking the phenotypic and proliferation markers as targets to identify novel cell proliferation pathways or cell cycle regulators. As a subset of morphologically distinct and non-disrupted PBCL completely lack the expression of tumor suppressors, phenotypic markers, and cell proliferation specific markers (Figure 7), it is likely that the growth and expansion of these cells are regulated by previously undescribed mechanisms or pathways [138,139]. Therefore, microdissection of these PBCL for gene expression profiling may lead to the identification of novel cell proliferation pathways and novel cell cycle regulators.
In summary, above findings and analyses strongly suggest that the PBCL is the most effective but largely ignored target for the early detection and intervention of human prostate cancer. It is apparent that the human tissue-derived basic research data may provide a more realistic roadmap that allows to observe the direct interactions among different cell types and to avoid the potential misleading from in vitro and animal studies.

Acknowledgments
All studies from Dr. Yan-gao Man's laboratory were financially supported in part by grants from All pictures presented in the current commentary were taken from the above studies on human prostate tissues with detailed clinic parameters and follow-up data from the Armed Forces Institute of Pathology and American Registry of Pathology, which was the world's largest and most comprehensive second-opinion consultation center with the world's largest human tissue repository. As the basic medical scientific studies are traditionally conducted on cancer cell lines or animal models, where it is impossible to completely mirror or mimic the human in vivo status, we sincerely hope that these pictures would serve as standards for defining the intrinsic value of the experimental findings from cell lines or cell line-derived animal models.